Wnt is a secreted protein that binds to surface receptors of neighboring cells to regulate the expression of various genes. The Wnt gene family was found ubiquitously in various types of tumors and was given a name “int” as the first cellular oncogene. Since then, it was found that a wingless gene implicated in the metamorphosis of Drosophila species is a homolog of the gene int-1 and therefore it was also suggested that the int gene will play a crucial role in the embryogenesis of Drosophila species. Accordingly, the name Wnt was coined as a combination of Wg (wingless) and Int.
The Wnt signaling pathway has been recognized as a key pathway responsible for oncogenesis. A member of the Wnt signaling pathway, such as adenomatous polyposis coli (APC), Axin, Axin2, βTrCP (E3 ligase), or the like, that binds to β-catenin to form an inactivated complex, may undergo mutagenesis incapable of degrading β-catenin and β-catenin may also be mutated to a non-degradable form, which leads to increased nuclear β-catenin accumulation, finally resulting in activation of the Wnt/β-catenin pathway. High nuclear accumulation of β-catenin leads to formation of a complex of β-catenin with T-cell factor/Lymphoid enhancing factor (TCF/LEF), which consequently results in induction of transcription of target genes.
Activation of the Wnt/β-catenin pathway is known to elicit regulated expression of numerous genes involved in carcinogenesis. The Wnt/β-catenin pathway induces expression of c-myc and cyclin D1 to thereby activate cell division, and also induces expression of a growth factor receptor, c-met and a growth factor, fibroblast growth factor 18 (FGF 18) to thereby increase cell proliferation. Further, it increases expression of anti-apoptotic proteins such as survivin protein as well as proteins necessary for cell proliferation, and induces expression of a vascular endothelial growth factor (VEGF) gene to stimulate tumor angiogenesis and provide a foundation for tumor growth and metastasis. In addition, for migration and metastasis of cancer cells, APC stimulated exchange factor (ASEF), matrix metalloproteinase (MMP) family, CD44 and the like, which are correlated with cell adhesion and extracellular matrix, function as target proteins of the Wnt/β-catenin pathway. A great deal of research and study has been actively attempted on development of an antitumor agent that is intended to target the Wnt/β-catenin pathway implicated in regulation of a variety of carcinogenesis-related proteins as described above. Unfortunately, most of such approaches made up to date are pathway inhibitors using chemical synthetic drugs which are designed based on a tertiary structure of individual proteins implicated in the Wnt/β-catenin pathway (Nick et al., Nature Reviews Drug Discovery, 5:997-1014, 2006).
RNA-mediated interference (RNAi) is a phenomenon wherein a 21-25 nucleotide-long double stranded siRNA specifically binds to a transcript (mRNA transcript) having a complementary sequence and degrades the corresponding transcript to thereby inhibit expression of a target protein of interest. As the RNA-mediated interference has recently suggested the solution to the problems encountered in development of conventional chemical synthetic drugs, many efforts have been made on development of various therapeutic agents, particularly antitumor agents, through selective inhibition of the expression of a certain protein at the transcript level. Production of target-directed small-molecule chemical drugs takes a long development period of time and tremendous development costs until they are optimized to certain protein targets, whereas the most pronounced advantage of siRNA drugs using the RNA-mediated interference phenomenon is in that it readily enables development of the optimized lead compounds for all the protein targets including non-druggable target substances. Protein or antibody drugs suffer from difficulties of production thereof due to complicated manufacturing processes, whereas siRNAs have significant advantages such as ease of synthesis, separation and purification, consequently relatively easy and convenient commercial-scale production, higher storage stability attributed to intrinsic nature of nucleic acid materials, as compared to protein drugs, and the like. Further, siRNA-based drugs are receiving a great deal of interest as a novel drug candidate group, based on a variety of strengths such as specific molecular target-directed antagonism, unlike conventional drugs (David et al., Nature Chemical Biology, 2:711-719, 2006).
The primary challenge associated with siRNA-based therapy is the identification of the optimum sequence where siRNA has the highest activity in the target base sequence. It is known that the efficiency of RNA-mediated interference is significantly affected by a specific binding site to the target transcript. Based on the database accumulated for the past several years, algorithms have been developed which are capable of designing a sequence position of siRNA substantially inhibiting expression of the target RNA, instead of simply binding to the transcript, and are currently available to users. However, it cannot be said that all of siRNAs determined by an in silico method using computer algorithms can effectively inhibit target RNAs in real cells and in vivo. Further, it is known that even when requirements necessary for complementary binding of siRNA to the target transcript are satisfied, the stability and intracellular location of RNAs and proteins, the state of proteins implicated in RNA-mediated interference, and a variety of other unknown factors are implicated in the determination of RNA-mediated interference efficiency. To this end, there is a need for development of a technique which will be carried out for a target protein by selecting several target sequence positions per transcript of one gene, preparing the corresponding siRNAs and screening an optimum position sequence having high expression-inhibitory activity from among such a candidate group (Derek et al., Annual Review of Biomedical Engineering, 8:377-402, 2006).